Purified thermostable Pyrococcus furiosus DNA polymerase I

Abstract

Purified thermostable Pyrococcus furiosus DNA polymerase that migrates on a non-denaturing polyacrylamide gel faster than phosphorylase B and Taq polymerase and more slowly than bovine serum albumin and has an estimated molecular weight of 90,000-93,000 daltons when compared with a Taq polymerase standard assigned a molecular weight of 94,000 daltons.

Description

CROSS REFERENCE TO RELATED APPLICATION [0001] This application is a continuation-in-part application of application Ser. No. 07 / 779,846, filed Oct. 18, 1991, which is a continuation-in-part of application Ser. No. 07 / 657,073, filed Feb. 19, 1991, which is a continuation-in-part of application Ser. No. 07 / 620,568, filed Dec. 3, 1990.TECHNICAL FIELD [0002] The present invention relates to a thermostable enzyme having DNA polymerase I activity useful in nucleic acid synthesis by primer extension reaction. BACKGROUND [0003] The archaebacteria are a recently discovered group of microorganisms that grow optimally at temperatures above 80° C. Some 20 species of these extremely thermophilic bacteria-like organisms have been isolated, mainly from shallow submarine and deep sea geothermal environments. Most of the archaebacteria are strict anaerobes and depend on the reduction of elemental sulfur for growth. [0004] The archaebacteria include a group of “hyperthermophiles” that grow optimally ...

AI Technical Summary

Benefits of technology

[0010] A thermostable DNA polymerase from the hyperthermophilic, marine archaebacterium, Pyrococcus furiosus (Pfu) has been discovered. The monomeric, multifunctional enzyme possesses both DNA polymerase and 3′ to 5′ exonuclease activities. The polymerase is extremely thermostable with a temperature optimum near 75° C. The purified enzyme functions effectively in the polymerase chain reaction (PCR). In addition, results from PCR fidelity studies indicate that Pyrococcus furiosus DNA polymerase yields amplification products containing 12 fold less mutations than reaction products from similar amplifications performed with Tag DNA polymerase. The 3′ to 5′ exonuclease dependent proofreading activity of Pfu DNA polymerase will excise mismatched 3′ terminal nucleotides from primer:template complexes and correctly incorporate nucleotides complementary to the template strand.

Problems solved by technology

However, knowledge of the metabolism of the hyperthermophilic microorganisms is presently very limited.

Although Tag polymerase is used in the vast majority of PCR performed today, it has a fundamental drawback: purified Taq DNA polymerase enzyme is devoid of 3′ to 5′ exonuclease activity and thus cannot excise misinserted nucleotides (Tindall, et al., Biochemistry, 29:5226-5231 (1990)).

Mutations introduced by Taq polymerase during DNA amplification have hindered PCR applications which require high fidelity DNA synthesis.

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